Project Summary Cell-wall targeting beta-lactam antibiotics are the largest group of antibacterial agents and have been the mainstay for treatment of bacterial infections. However, most beta-lactam antibiotics cannot be used for the treatment of methicillin-resistant Staphylococcus aureus (MRSA) infections because MRSA produces a cell- wall synthesis enzyme PBP2A, which most beta-lactams cannot inactivate. Although PBP2A is the primary determinant of MRSA beta-lactam resistance, the overall beta-lactam resistance of MRSA is affected by additional bacterial factors such as FtsH, a protease in the cell membrane. Overexpression of FtsH makes MRSA sensitive specifically to beta-lactams. The beta-lactam-sensitizing effect of FtsH is due to its degradation of YpfP, an enzyme synthesizing the anchor molecule for lipoteichoic acid (LTA). The YpfP- degradation by FtsH causes the production of abnormally large LTA. On the other hand, increased production of normal LTA by deletion of the ftsH gene makes MRSA more resistant to beta-lactams. When the production of normal LTA is suppressed by inhibiting the production of the LTA synthesis enzyme LtaS, MRSA becomes hypersensitive to beta-lactams. Based on these results, this study hypothesizes that the large LTA lowers MRSA beta-lactam resistance by suppressing the enzyme activities of the cell-wall synthesis enzymes whereas the normal LTA is required for them. Even under the beta-lactam sensitizing conditions, MRSA can regain resistance to beta-lactams. Genome-sequencing of 26 such mutants showed that 16 genes are critical for MRSA beta-lactam resistance. The long-term goal of this project is to develop novel ?-lactam potentiators against MRSA. The objective of this study is to understand how FtsH and the disturbance in LTA synthesis sensitize MRSA to ?-lactams, and how MRSA regains ?-lactam resistance. In aim 1, with the MRSA strains producing either the large LTA or reduced amount of the normal LTA, the cell wall synthesis steps affected by the LTA molecules will be determined. The effect of LTA on cell wall synthesis will be directly tested by a peptidoglycan-synthesis assay. Also, the bacterial two-hybrid analysis will determine whether the degradation of YpfP is modulated by FtsH-binding proteins. In aim 2, by generating each of the resistance mutations in the chromosome of MRSA, the mutations critical for MRSA ?-lactam resistance will be determined. Also, transposon-mediated mutagenesis will identify the genes essential for MRSA to regain the ?-lactam resistance. Finally, to test whether the roles of the 16 genes are universally conserved among S. aureus strains, resistant mutants will be identified from two more MRSA strains, and their genomes will be sequenced. Completion of this study will reveal the intricate network of the beta-lactam resistance regulators in MRSA and open the door to developing novel beta-lactam potentiators with minimal risk of resistance development in the treatment of MRSA infections. Such drugs are expected to reduce the burdens of antibiotic resistance.